Reagents and methods for detecting protein lysine 2-hydroxyisobutyrylation

ABSTRACT

The invention provides an isolated peptide comprising a lysine 2-hydroxyisobutyrylation site, a lysine 2-hydroxyisobutyrylation specific affinity reagent that specifically binds to the peptide, and a method for detecting protein lysine 2-hydroxyisobutyrylation in a sample using the reagent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/941,973, filed on Jul. 15, 2013, and the contents of which areincorporated by reference herein, in their entireties and for allpurposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.DK082664 and RR020389 awarded by the National Institute of Health. TheUnited States has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to reagents and methods for detecting proteinshaving post-translational modifications. More particularly, it relatesto peptides comprising a 2-hydroxyisobutyrylated lysine, and their usesto develop reagents and methods useful for detecting protein lysine2-hydroxyisobutyrylation.

BACKGROUND OF THE INVENTION

Chromatin is decorated with a wide variety of protein post-translationalmodifications (PTMs), such as acetylation, methylation, phosphorylationand recently β-N-acetylglucosamine (O-GlcNAc) modifications. Theregulatory potential of histone marks is well illustrated for histonelysine acetylation (K_(ac)) and lysine methylation. Small chemicaldifferences at a given lysine residue, e.g., acetylation vs methylation,may associate with very different functional outputs. Moreover, subtledifferences within the same family of modifications can be of functionalsignificance. For instance, lysine methylation can be present in threeforms: mono-, di- or tri-methylation. Depending on the methylation stateand its position, histone lysine methylation can be involved in eitherthe activation or repression of gene expression. For example, lysinemonomethylation and lysine trimethylation of histone H3K4 respectivelymark enhancers and promoters of active genes. The dysregulation ofhistone marks can lead to diverse diseases, such as cancer.

The differentiation of male germ cells offers a particularly interestingsetting to explore the biological significance of new histone PTM.Indeed, during this process, the male genome undergoes severallarge-scale structural and functional changes, including theestablishment of stage-specific expression patterns, and a genome-widereplacement of histones by transition proteins and protamines.

There remains a need for developing reagents and methods useful fordetecting post-translational modifications of histones or nonhistoneproteins linked to various diseases and disorders.

SUMMARY OF THE INVENTION

The present invention relates to the use of peptides comprising a2-hydroxyisobutyrylated lysine (K_(2ohibu)) to develop reagents andmethods for detecting protein lysine 2-hydroxyisobutyrylation,especially site specific lysine 2-hydroxyisobutyrylation.

An isolated peptide comprising a 2-hydroxyisobutyrylated lysine isprovided. The isolated peptide may be derived from a histone protein ora fragment thereof. The histone protein may be derived from an organismselected from the group consisting of human, mouse, S. cerevisiae,Tetrahymena thermophila, D. melanogaster, and C. elegans. The isolatedpeptide may comprise an amino acid sequence having at least 70% identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 29-112. The isolated peptide may comprise an amino acid sequenceselected from SEQ ID NOs: 29-112. The isolated peptide may comprise atleast 2 amino acid residues on each of the N-terminal and C-terminalsides of the 2-hydroxyisobutyrylated lysine.

An isolated lysine 2-hydroxyisobutyrylation specific affinity reagent isalso provided. It is capable of binding specifically to a peptidecomprising a 2-hydroxyisobutyrylated lysine. The peptide may comprise anamino acid sequence selected from SEQ ID NOs: 29-112. The binding may bedependent on the presence of the 2-hydroxyisobutyrylated lysine but nota surrounding peptide sequence thereof in the peptide. The binding maybe dependent on the presence of the 2-hydroxyisobutyrylated lysine and asurrounding peptide sequence thereof in the peptide. The lysine2-hydroxyisobutyrylation specific affinity reagent may be a protein oran antibody.

A method for producing a lysine 2-hydroxyisobutyrylation specificaffinity reagent that is a protein is provided. The method comprisesscreening a protein library using a peptide comprising a2-hydroxyisobutyrylated lysine and at least two amino acid residues oneach of the N-terminal and C-terminal sides of the2-hydroxyisobutyrylated lysine. The protein library may be selected fromthe group consisting of a phage display library, a yeast displaylibrary, a bacterial display library, and a ribosome display library.

A method for producing a lysine 2-hydroxyisobutyrylation specificaffinity reagent that is an antibody is also provided. The methodcomprises immunizing a host with a peptide comprising a2-hydroxyisobutyrylated lysine and at least two amino acid residues oneach of the N-terminal and C-terminal sides of the2-hydroxyisobutyrylated lysine.

A method for detecting a 2-hydroxyisobutyrylated lysine in a protein ora fragment thereof is provided. The method comprises contacting theprotein or a fragment thereof with the isolated lysine2-hydroxyisobutyrylation specific affinity reagent capable of bindingspecifically to a peptide comprising a 2-hydroxyisobutyrylated lysine.The lysine 2-hydroxyisobutyrylation specific affinity reagent and theprotein or a fragment thereof forms a binding complex. The methodfurther comprise detecting the binding complex. The presence of thebinding complex indicates the presence of a 2-hydroxyisobutyrylatedlysine in the protein or a fragment thereof. In this method, the lysine2-hydroxyisobutyrylation specific affinity reagent may be a protein oran antibody.

A method for determining the level of protein lysine2-hydroxyisobutyrylation in a sample is provided. The method comprisesdetecting a 2-hydroxyisobutyrylated lysine in the sample.

A kit for detecting a 2-hydroxyisobutyrylated lysine in a protein of afragment thereof is provided. The kit comprises an isolated lysine2-hydroxyisobutyrylation specific affinity reagent capable of bindingspecifically to a peptide comprising a 2-hydroxyisobutyrylated lysine.

A kit for isolating a peptide containing a 2-hydroxyisobutyrylatedlysine is also provided. The kit comprises an isolated lysine2-hydroxyisobutyrylation specific affinity reagent capable of bindingspecifically to a peptide comprising a 2-hydroxyisobutyrylated lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows chemical structures of lysine, acetyllysine, and otherpossible chemical structures of lysine modifications that may have theelemental composition of C₄H₇O₂ and induce a mass shift of +86.0368 Da.

FIG. 2 shows verification of 2-hydroxyisobutyrylation at histone H4K77.(A) The MS/MS spectra of an in vivo peptide bearing a PTM(DAVTYTEHAK_(+86.0354)R) (DAVTYTEHAKR; SEQ ID NO: 29) (top), a syntheticlysine 2-hydroxyisobutyrylated peptide corresponding to the sequence ofthe in vivo peptide (middle), and a mixture of the two peptides(bottom). The label A designates b or y ions with water and/or ammonialoss. Insets show the precursor ion masses. (B) Extracted ionchromatograms (XICs) of a mixture of four synthetic peptides(DAVTYTEHAK_((±)-2ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29),DAVTYTEHAK_((r)-3ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29),DAVTYTEHAK_((s)-3ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29) andDAVTYTEHAK_(4ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29)) and the in vivopeptide. (C) Extracted ion chromatograms (XIC) of the in vivo peptide(top), the synthetic peptide DAVTYTEHAK_(2ohibu)R (DAVTYTEHAKR; SEQ IDNO: 29) (middle), and the mixture of the both peptides (bottom).

FIG. 3 shows histone K_(2ohibu) in eukaryotic cells. (A) Westernblotting analysis of the recombinant H4 and HeLa cell core histones.Immunoblot against H4 was used as a loading control. (B) Detection ofK_(2ohibu) and H4K8_(2ohibu) in human HeLa cells, MEF cells, DrosophilaS2 cells and yeast S. cerevisiae cells by Western blotting. (C) TheK_(2ohibu) sites from lysine 34 to lysine 120 in histone H2B shown in 3Dstructure of mouse nucleosome. H2B in the nucleosome is in ribbon shapeand the other histones are displayed as backbones. The K_(2ohibu)residues are highlighted in molecular ball shapes. (D) The lysineresidues of all the K_(2ohibu) sites in human and mouse histones areshown in red and bold: H1.2 corresponding to residues 2-212 of H12_MOUSE(SEQ ID NO: 6), H2A corresponding to residues 2-130 of H2A.1_HUMAN (SEQID NO: 2), H2B corresponding to residues 2-126 of H2B.1B-HUMAN (SEQ IDNO: 3), H3 corresponding to residues 2-136 of H33_HUMAN (SEQ ID NO: 4)and H4 corresponding to residues 2-103 of H4_HUMAN (SEQ ID NO: 5). Theunique sites with K_(2ohibu), but without K_(cr) and K_(ac) beingreported are labeled with underscore. To make a comparison, the knownK_(ac) sites (labeled with a black triangle) and K_(cr) sites (labeledwith a green star) described in the literature were also listed.

FIG. 4 shows spatiotemporal analyses of H4K8_(2ohibu) and H4K8_(ac) inmouse sperm cells. Sections from paraffin embedded testes were used forthe detection of H4K8_(2ohibu) (A) and H4K8_(ac) (B) byimmunohistochemistry (IH), with or without counterstaining. The tubulestages are indicated above each panel. The insets show a highermagnification of a dot-like structure observed in round spermatids. (C)Both H4K8_(2ohibu) and H4K8_(ac) marks are visualized byimmunofluorescence (IF) in round spermatids from seminiferous tubulepreparations. In each panel, the indicated PTMs (green) are co-detectedalong with HP1gamma (red).

FIG. 5 shows high-Resolution mapping of H4K8_(2ohibu) and H4K8_(ac) inspermatocytes (meiotic cells) and round spermatids (post-meiotic cells).(A) The upper panels show the distributions of H4K8_(2ohibu) (right) andH4K8_(ac) (left) peaks between the two cell populations (spermatocytes(Spc) and round spermatids (RS)), while the lower panels show thedistributions of overlapped and unique peaks for H4K8_(2ohibu) andH4K8_(ac) in Spc (left) and RS (right). (B) Metagene analysis ofH4K8_(2ohibu) and H4K8_(ac) peaks with respect to their associatedgenes' transcription starting site (TSS). Color code for cell types andthe histone marks are indicated. TES means transcription end site. (C)and (D) Distributions per chromosome of H4K8_(ac) peaks (C) andH4K8_(2ohibu) peaks (D). Peaks observed in spermatocytes and spermatidsare represented in blue and red, respectively. The sex chromosomes arehighlighted to emphasize the evolution of peak intensities on thesechromosomes between spermatocytes and round spermatids.

FIG. 6 shows that H4K8_(2ohibu) is associated with genes with hightranscriptional activity in male germ cells. (A) Number of genesassociated with H4K8_(2ohibu) and/or H4K8_(ac) peaks in male germ cells(spermatocytes and/or round spermatids). (B) Expression of genesassociated with H4K8_(2ohibu) peaks in mouse tissues: distribution ofH4K8_(2ohibu) associated genes according to their sites of predominantexpression in mouse tissues. (C) Gene expression patterns oftestis-specific genes associated with H4K8_(2ohibu) in spermatogeniccells: the heatmap shows the expression levels of H4K8_(2ohibu)associated genes with a testis predominant expression in meiotic cells(spermatocytes: Spc) and post-meiotic cells (round spermatids: RS).Color scale: from green (low expression) to red (high expression). (D)Box plots comparing the distribution of expression levels of genesassociated with H4K8_(2ohibu) and/or H4K8_(ac) peaks in meiotic cells(Spermatocytes: Spc) and in post-meiotic cells (Round spermatids: RS).*p<0.001 (unpaired t-test) showing a significant difference with thereference group of genes (not associated with H4K8_(2ohibu) or H4K8_(ac)peaks); **p<0.001 (unpaired t-test) showing a significant differencewith all the other groups of genes including the reference group ofgenes (not associated with H4K8_(2ohibu) or H4K8_(ac) peaks) and thegenes associated with either H4K8_(ac) or H4K8_(2ohibu) alone. (E)Proportions of X-linked genes and autosomal genes associated withH4K8_(2ohibu) (left panel) or H4K8_(ac) (right panel).

FIG. 7 shows protein sequences of human histone proteins (A) H1.2 (SEQID NO: 1), (B) H2A (SEQ ID NO: 2), (C) H2B (SEQ ID NO: 3), (D) H3 (SEQID NO: 4), and (E) H4 (SEQ ID NO. 5).

FIG. 8 shows protein sequences of mouse histone proteins (A) H1.2 (SEQID NO: 6), (B) H2A (SEQ ID NO: 7), (C) H2B (SEQ ID NO: 8), (D) H3 (SEQID NO: 9), and (E) H4 (SEQ ID NO: 10).

FIG. 9 shows protein sequences of S. cerevisiae histone proteins (A) H2A(SEQ ID NO: 11), (B) H2B (SEQ ID NO: 12), (C) H3 (SEQ ID NO: 13), and(D) H4 (SEQ ID NO: 14).

FIG. 10 shows protein sequences of Tetrahymena histone proteins (A) H2A(SEQ ID NO: 15), (B) H2B (SEQ ID NO: 16), (C) H3 (SEQ ID NO: 17), and(D) H4 (SEQ ID NO: 18).

FIG. 11 shows protein sequences of D. melanogaster histone proteins (A)H1 (SEQ ID NO: 19), (B) H2A (SEQ ID NO: 20), (C) H2B (SEQ ID NO: 21),(D) H3 (SEQ ID NO: 22), and (E) H4 (SEQ ID NO: 23).

FIG. 12 shows protein sequences of c. elegans histone proteins (A) H1(SEQ ID NO: 24), (B) H2A (SEQ ID NO: 25), (C) H2B (SEQ ID NO: 26), (D)H3 (SEQ ID NO: 27), and (E) H4 (SEQ ID NO: 28).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a new type of histonemarks, lysine 2-hydroxyisobutyrylation. In particular, over 60 novelhistone lysine 2-hydroxyisobutyrylation sites in human and mouse cellshave been identified. Genome-wide mapping of histone H4 lysine 82-hydroxyisobutyrylation (H4K8_(2ohibu)), in parallel with histone H4lysine 8 acetylation (H4K8_(ac)), in spermatogenic cells, shows thatH4K8_(2ohibu) is a new indicator of gene transcriptional activity,demonstrating that lysine 2-hydroxyisobutyrylation represents a novelactive histone mark with important functions in the physiologicalsetting of male germ cell differentiation. Peptides derived from histoneproteins or fragments thereof comprising a K_(2ohibu) site may be usedto generate reagents useful for detecting protein lysine2-hydroxyisobutyrylation, especially for detecting site specific proteinlysine 2-hydroxyisobutyrylation.

The term “peptide” used herein refers to a linear chain of two or moreamino acids linked by peptide bonds. A peptide may have about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40,50, 100, 200 or more amino acids. The amino acids of a peptide may bemodified, deleted, added or substituted. A peptide may be obtained usingconventional techniques known in the art. For example, a peptide may besynthesized or obtained from a native or recombinant protein byenzymatic digestion.

The term “polypeptide” used herein refers to a peptide having at least 4amino acids, preferably at least about 20 amino acids, regardless ofpost-translational modification. The term “protein” used herein refersto a biological molecule consisting of one or more polypeptides,regardless of post-translational modification. Each polypeptide in aprotein may be a subunit. The polypeptide or protein may be in a nativeor modified form, and may exhibit a biological function orcharacteristics.

Where a protein is a single polypeptide, the terms “protein” and“polypeptide” are used herein interchangeably. A fragment of apolypeptide or protein refers to a portion of the polypeptide or proteinhaving an amino acid sequence that is the same as a part, but not all,of the amino acid sequence of the polypeptide or protein. Preferably, afragment of a polypeptide or protein exhibits a biological function orcharacteristics identical or similar to that of the polypeptide orprotein.

The term “derived from” used herein refers to the origin or source fromwhich a biological molecule is obtained, and may include naturallyoccurring, recombinant, unpurified or purified molecules. A biologicalmolecule such as a peptide (e.g., a polypeptide or protein) may bederived from an original molecule, becoming identical to the originalmolecule or a variant of the original molecule. For example, a peptidederived from an original peptide may have an amino acid sequenceidentical or similar to the amino acid sequence of its original peptide,with at least one amino acid modified, deleted, inserted, orsubstituted. A derived peptide may have an amino acid sequence at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%,preferably at least about 50%, more preferably at least about 80%, mostpreferably at least about 90%, identical to the amino acid sequence ofits original peptide, regardless of post-translational modification.Preferably, a derived biological molecule (e.g., a peptide) may exhibita biological function or characteristics identical or similar to that ofthe original biological molecule.

The term “antibody” used herein includes whole antibodies, and antigenbinding fragments (or antigen-binding portions) and single chainsthereof. A whole antibody can be either one of the two types. The firsttype refers to a glycoprotein typically having two heavy chains and twolight chains, and includes an antigen binding portion. For example, theantibody may be a polyclonal or monoclonal antibody. The term “antigenbinding portion” of an antibody used herein refers to one or morefragments of the antibody that retain the ability of specificallybinding to an antigen. The second type refers to a heavy-chain antibodyoccurring in camelids that is also called Nanobody. The term“single-chain variable fragment” of an antibody used herein refers to afusion protein of the variable regions of the heavy and light chains ofthe antibody, connected with a short linker peptide, for example, ofabout 20-25 amino acids, that retains the ability of specificallybinding to an antigen.

An isolated peptide comprising a 2-hydroxyisobutyrylated lysine isprovided. The term “2-hydroxyisobutyrylated lysine” used herein refersto a lysine residue that is 2-hydroxyisobutyrylated on its epsilon-aminegroup. It is also known as a 2-hydroxyisobutyryl lysine residue. Theterm “lysine 2-hydroxyisobutyrylation site” used herein refers to alysine residue in a peptide, polypeptide or protein that may be2-hydroxyisobutyrylated on the epsilon-amine group of the lysineresidue. The term “lysine 2-hydroxyisobutyrylation” used herein refersto 2-hydroxyisobutyrylation on the epsilon-amine group of a lysineresidue that generates a 2-hydroxyisobutyryl lysine residue or2-hydroxyisobutyrylated lysine.

The peptide of the present invention may have at least about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,80, 90, 100, 150 or 200 amino acids. The peptide may have about 3-25amino acids, preferably 5-20 amino acids, more preferably 6-14 aminoacids.

The peptide of the present invention may be prepared using conventionaltechniques known in the art. The peptide may be derived from a protein,for example, a histone protein, or a fragment thereof, having a lysine2-hydroxyisobutyrylation site. The histone protein may be derived from aeukaryotic cell. Examples of a eukaryotic cell include cells from ayeast (e.g., S. cerevisiae), an C. elegans, a Drosophila (e.g., D.melanogaster (S2)), a Tetrahymena (e.g., Tetrahymena thermophila), amouse (e.g., M. musculus (MEF)), or a human. Preferably, the eukaryoticcell is a mammalian cell, for example, a human, primate, mouse, rat,horse, cow, pig, sheep, goat, chicken, dog or cat cell. More preferably,the eukaryotic cell is a human cell.

The histone protein may be a histone linker protein or a histone coreprotein. A histone linker protein may be selected from the members ofthe H1 family, including the H1F subfamily (e.g., H1F0, H1FNT, H1FOO,and H1FX) and the H1H1 subfamily (e.g., HIST1H1A, HIST1H1B, HISTIHIC,HIST1H1D, HIST1HIE and HIST1H1T). A histone core protein may a member ofthe H2A, H2B, H3 or H4 family. A histone core protein in the H2A familymay be a member of the H2AF subfamily (e.g., H2AFB1, H2AFB2, H2AFB3,H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, and H2AFZ), the H2A1 subfamily(e.g., HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG,HIST1H2AH, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, and HIST1H2AM),or the H2A2 subfamily (e.g., HIST2H2AA3, HIST2H2AA4, HIST2H2AB, andHIST2H2AC). A histone core protein in the H2B family may be a member ofthe H2BF subfamily (e.g., H2BFM and H2BFWT), the H2B1 subfamily (e.g.,HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF,HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL,HIST1H2BM, HIST1H2BN, and HIST1H2BO), or the H2B2 subfamily (e.g.,HIST2H2BE and HIST2H2BF). A histone core protein in the H3 family may bea member of the H3A1 subfamily (e.g., HIST1H3A, HIST1H3B, HIST1H3C,HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, andHIST1H3J), the H3A2 subfamily (e.g., HIST2H3A, HIST2H3C, and HIST2H3D),or the H3A3 subfamily (e.g., HIST3H3), the H3A3 subfamily (e.g., H3F3A,H3F3B, and H3F3C). A histone core protein in the H4 family may be amember of the H41 subfamily (e.g., HIST1H4A, HIST1H4B, HIST1H4C,HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J,HIST1H4K, and HIST1H4L), or the H44 subfamily (e.g., HIST4H4).

The protein and gene sequences of histone proteins in various speciesare known in the art. For example, protein sequences of human H1.2, H2A,H2B, H3 and H4 histone proteins can be found in GenBank databaseAccession Nos. P16403 (H12_HUMAN) having SEQ ID NO: 1 (FIG. 7A), P04908(H2A1B_HUMAN) having SEQ ID NO: 2 (FIG. 7B), P33778 (H2B1B_HUMAN) havingSEQ ID NO: 3 (FIG. 7C), P84243 (H33_HUMAN) having SEQ ID NO: 4 (FIG. 7D)and P62805 (H4 HUMAN) having SEQ ID NO: 5 (FIG. 7E) respectively. Theprotein sequences of mouse histone proteins H1.2, H2A, H2B, H3 and H4can be found in the GenBank database Accession Nos. P15864 (H12_MOUSE)having SEQ ID NO: 6 (FIG. 8A), P22752 (H2A1_MOUSE) having SEQ ID NO: 7(FIG. 8B), Q64475 (H2B1B_MOUSE) having SEQ ID NO: 8 (FIG. 8C), P84244(H33_MOUSE) having SEQ ID NO: 9 (FIG. 8D) and P62806 (H4_MOUSE) havingSEQ ID NO: 10 (FIG. 8E), respectively. Histone protein sequences ofother species such as S. cerevisiae (FIG. 9), Tetrahymena (FIG. 10), D.melanogaster (FIG. 11), and C. elegans (FIG. 12) are also known.

A fragment of a histone protein may have an amino acid sequence that isthe same as a part, not all, of the amino acid sequence of the histoneprotein comprising at least one lysine 2-hydroxyisobutyrylation site.The histone protein fragment may have at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,80, 90, 100, 150 or 200 amino acids. The histone fragment may have about3-25 contiguous amino acids, preferably about 5-20 contiguous aminoacids, more preferably about 6-14 contiguous amino acids, of the histoneprotein covering at least one lysine 2-hydroxyisobutyrylation site inthe histone protein.

The histone protein or fragment may have a 2-hydroxyisobutyrylatedlysine at a lysine 2-hydroxyisobutyrylation site. The lysine2-hydroxyisobutyrylation site may be any one of the lysine2-hydroxyisobutyrylation sites in exemplary histone proteins of human(Table 1), mouse (Table 2), S. cerevisiae (Table 3), and Tetrahymena(Table 4).

A histone protein may be obtained from a biological sample or preparedusing recombinant techniques. A histone protein fragment may be preparedby recombinant techniques, or by digesting the histone protein with anenzyme (e.g., trypsin). The lysine 2-hydroxyisobutyrylation site in thehistone protein or fragment may be lysine 2-hydroxyisobutyrylatednaturally or artificially. The presence of a 2-hydroxyisobutyrylatedlysine may be confirmed by using conventional techniques known in theart, for example, mass spectrometry.

The peptide of the present invention may comprise an amino acid sequencehaving at least about 70%, 80%, 90%, 95% or 99%, preferably at leastabout 90%, more preferably 100%, identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29-112. The peptidemay encompass any lysine 2-hydroxyisobutyrylation site with or withoutits surrounding sequences from a histone proteins. The peptide maycomprise more than one 2-hydroxyisobutyrylated lysine. The peptide mayalso comprise a protein post-translational modification other than2-hydroxyisobutyrylated lysine, such as acetylated lysine or methylatedlysine. The peptides may further comprise at least about 1, 2, 3, 4, 5,5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal andC-terminal sides of the 2-hydroxyisobutyrylated lysine. Preferably, thepeptide may comprise at least 2 amino acid residues on each of theN-terminal and C-terminal side of the 2-hydroxyisobutyrylated lysine.Exemplary peptides of the present invention are shown in Tables 1-4.

An isolated lysine 2-hydroxyisobutyrylation specific affinity reagent isalso provided. The term “lysine 2-hydroxyisobutyrylation specificaffinity reagent” used herein refers to a molecule that is capable ofbinding to a peptide, polypeptide or protein having a lysine2-hydroxyisobutyrylation site, which may be a histone protein or apeptide of the present invention. The lysine 2-hydroxyisobutyrylationspecific affinity reagent may be a protein, for example, an antibody.The lysine 2-hydroxyisobutyrylation site may be any lysine2-hydroxyisobutyrylation site in any histone protein from any species.Examples of the lysine 2-hydroxyisobutyrylation sites include those inhuman (Table 1), mouse (Table 2), S. cerevisiae (Table 3), andTetrahymena(Table 4), and homologous lysine sites in correspondingeukaryotic histone proteins.

In some embodiments, the lysine 2-hydroxyisobutyrylation specificaffinity reagent binds a peptide, polypeptide or protein having a lysine2-hydroxyisobutyrylation site that is 2-hydroxyisobutyrylated, having anaffinity that is at least about 10, 50, 100, 500, 1000 or 5000 timeshigher than that for its counterpart when the site is not2-hydroxyisobutyrylated.

In other embodiments, the lysine 2-hydroxyisobutyrylation specificaffinity reagent binds a peptide, polypeptide or protein having a lysine2-hydroxyisobutyrylation site that is not 2-hydroxyisobutyrylated,having an affinity that is at least about 10, 50, 100, 500, 1000 or 5000times higher than that for its counterpart when the site is2-hydroxyisobutyrylated. The lysine 2-hydroxyisobutyrylation specificaffinity reagent may be a peptide, polypeptide or protein, which may bean antibody. Preferably, the peptide is a peptide of the presentinvention.

The lysine 2-hydroxyisobutyrylation specific affinity reagent may besite specific, i.e., the binding is dependent on the presence of the2-hydroxyisobutyrylated lysine and its surrounding peptide sequence. Thesurrounding peptide sequence may include at least about 1, 2, 3, 4, 5,5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal andC-terminal sides of the 2-hydroxyisobutyrylated lysine. For example, thebinding depends on the presence of the 2-hydroxyisobutyrylated lysineand at least 2 amino acid residues on each of the N-terminal andC-terminal side of the 2-hydroxyisobutyrylated lysine.

The lysine 2-hydroxyisobutyrylation specific affinity reagent may not besite specific, i.e., the binding is dependent on the presence of the2-hydroxyisobutyrylated lysine but not its surrounding peptide sequence.One example is an anti-lysine-2-hydroxyisobutyrylation pan antibody.

A method for producing the lysine 2-hydroxyisobutyrylation specificaffinity reagent of the present invention is further provided.

Where the lysine 2-hydroxyisobutyrylation specific affinity reagent is aprotein, the protein may be produced by screening a protein library(also known as a display library or a degenerated protein library) usingthe peptide of the present invention. The peptide may have at least twoamino acid residues one each of the N-terminal and C-terminal sides ofthe 2-hydroxyisobutyrylated lysine. The protein library may consist ofmany degenerated protein sequences, which may comprise two regions: oneor more fixed peptide sequence regions and a plurality of degeneratedamino acid sequences. The protein library may be a phage proteinlibrary, a yeast protein library, bacterial protein library, ribosomeprotein library, or other synthetic protein library comprising peptideshaving randomized amino acid sequences.

Where the lysine 2-hydroxyisobutyrylation specific affinity reagent isan antibody, the antibody may be produced by different methods known inthe art. For example, the production method may comprise immunizing ahost with an antigenic peptide to produce the antibody. The method mayfurther comprise collecting antisera from the host. The host may be amammal suitable for producing antibodies. For example, the host may be amouse, rabbit, goat, Camelidae family animal (such as Lama and camel),or cartilaginous fishes. Dependent on the host used, the generatedantibody can contain either two chains (a heavy chain and a light chain)or one chain (or heavy chain-only antibody occurring in camelids) thatis also called Nanobody.

The antigenic peptide may be derived from a histone protein or afragment thereof comprising a lysine 2-hydroxyisobutyrylation site,which may be 2-hydroxyisobutyrylated or not. The antigenic peptide maycomprise a peptide of the present invention. Examples of antigenicpeptides having 2-hydroxyisobutyrylated lysine may comprise one or moreof the peptides in Tables 1 and 2. Examples of antigenic peptides nothaving 2-hydroxyisobutyrylated lysine may have an amino acid sequenceidentical to those in Tables 1 and 2, except that the lysine2-hydroxyisobutyrylation site is not 2-hydroxyisobutyrylated. TheN-terminal or C-terminal end of any of these peptides may be extended by1-20 residues.

The method may further comprise purifying the antibody from theantisera. The method may further comprise utilizing spleen cells fromthe host to generate a monoclonal antibody. In some embodiments, theantibody specifically binds to a histone protein or fragment having alysine 2-hydroxyisobutyrylation site when the site is2-hydroxyisobutyrylated, but not when the site is not2-hydroxyisobutyrylated. In other embodiments, the antibody specificallybinds to a histone protein or fragment having a lysine2-hydroxyisobutyrylation site when the site is not2-hydroxyisobutyrylated, but not when the site is2-hydroxyisobutyrylated.

The method may further comprise deduce the antibody sequences byhigh-performance liquid chromatography (HPLC)-mass spectrometry analysisof the isolated antibodies and followed by protein sequence databasesearch against all the possible IgG protein sequences (derived from cDNAsequences) from bone marrow (or B cells) of the immunized host. The IgGcDNA sequences can be obtained from conventional DNA sequencingtechnologies from IgG cDNAs that are generated by RT-PCR using the knownart. The derived heavy- and light-chain variable regions (VH and VL) canbe further paired (in case the IgG is from a two-chain antibodies from ahost like mice or rabbit). Such a pairing is not necessary for those IgGderived from heavy chain-only antibody (or Nonabody) from Lama. Theantibody can then be generated using the antibody sequence informationusing the known art.

A method for detecting a 2-hydroxyisobutyrylated lysine in a protein orits fragment is provided. The method comprises (a) contacting theprotein or its fragment with a lysine 2-hydroxyisobutyrylation specificaffinity reagent of the present invention to form a binding complex, and(b) detecting the binding complex. The presence of the binding complexindicates the presence of the 2-hydroxyisobutyrylated lysine in theprotein or its fragment. The binding complex may be detected by usingvarious conventional methods in the art. The protein may be a histoneprotein. The method may further comprise quantifying the amount of thebinding complex. The amount of the binding complex may indicate thelevel of lysine 2-hydroxyisobutyrylation in the protein or its fragment.

For each detection method of the present invention, a kit is provided.The kit comprises a lysine 2-hydroxyisobutyrylation specific affinityreagent of the present invention. The kit may further comprise aninstruction directing how to carry out the method.

A fusion protein reporter is provided. The fusion protein reportercomprises a core flanked by a donor fluorescent moiety and an acceptorfluorescent moiety. The core includes a peptide, which comprises alysine 2-hydroxyisobutyrylation site and a lysine2-hydroxyisobutyrylation binding domain. The term “lysine2-hydroxyisobutyrylation binding domain” used herein refers to a regionin a protein sequence capable of specific binding to the lysine2-hydroxyisobutyrylation site.

The fusion protein reporter of the present invention may be useful fordetermining protein lysine 2-hydroxyisobutyrylation level in a sample orscreening for an agent that regulates protein lysine2-hydroxyisobutyrylation by using the fluorescence resonance energytransfer (FRET). The FRET involves the transfer of photonic energybetween fluorophores when in close proximity. Donor fluorescent moietiesand acceptor fluorescent moieties suitable for FRET are known in theart. In the fusion protein reporter, the donor fluorescent moiety may beselected from the group consisting of cyan fluorescent protein (CFP),enhanced cyan fluorescent protein (ECFP), and A206K mutants thereof, andthe acceptor fluorescent moiety may be selected from the groupconsisting of yellow fluorescent protein (YFP), enhanced yellowfluorescence protein (EYFP), Citrine, Venus, and A206K mutants thereof.

The peptide in the fusion protein reporter may comprise a peptide of thepresent invention. It may be derived from a histone protein or fragmentcomprising a lysine 2-hydroxyisobutyrylation site, where the histoneprotein or fragment may be 2-hydroxyisobutyrylated or not at the lysine2-hydroxyisobutyrylation site.

The lysine 2-hydroxyisobutyrylation site may be located in theN-terminus, C-terminus or the core region of a histone protein. TheN-terminus, C-terminus, and core regions of histone proteins (e.g.,human or mouse H1.2, H2A, H2B, H3 or H4) are known in the art.

The fusion protein reporter may comprise one or more lysine2-hydroxyisobutyrylation binding domains. A lysine2-hydroxyisobutyrylation binding domain may be derived from a lysine2-hydroxyisobutyrylation specific affinity reagent of the presentinvention.

In some embodiments, the lysine 2-hydroxyisobutyrylation site in thepeptide is not 2-hydroxyisobutyrylated, and the lysine2-hydroxyisobutyrylation binding domain specifically binds to the lysine2-hydroxyisobutyrylation site when the site is 2-hydroxyisobutyrylated,but not when the sites is not 2-hydroxyisobutyrylated.

In other embodiments, the lysine 2-hydroxyisobutyrylation site in thepeptide is 2-hydroxyisobutyrylated, and the lysine2-hydroxyisobutyrylation binding domain specifically binds to the lysine2-hydroxyisobutyrylation site when the peptide is not lysine2-hydroxyisobutyrylated, but not when the site is2-hydroxyisobutyrylated.

The lysine 2-hydroxyisobutyrylation site may be conjugated to the lysine2-hydroxyisobutyrylation binding domain with a linker molecule. Thelinker molecule may be a peptide have any amino acid sequence, and mayhave about 1-50 amino acids, preferably 1-30 amino acids, morepreferably 2-15. In some embodiments, the linker molecule may be-Gly-Gly-. The length and contents of a linker molecule may be adjustedto optimize potential fluorescence resonance energy transfer (FRET)between the donor fluorescent moiety and the acceptor fluorescent moietywhen the lysine 2-hydroxyisobutyrylation site in the fusion proteinreporter is 2-hydroxyisobutyrylated or not, and bound by the lysine2-hydroxyisobutyrylating binding domain.

The fusion protein reporter may further comprise a targetingpolypeptide. The targeting polypeptide may be selected from the groupconsisting of a receptor ligand, a nuclear localization sequence (NLS),a nuclear export signal (NES), a plasma membrane targeting signal, ahistone binding protein, and a nuclear protein.

A method for determining the level of protein lysine2-hydroxyisobutyrylation in a sample. The method comprises detecting a2-hydroxyisobutyrylated lysine in the sample. The method may comprise(a) contacting the sample with a fusion protein reporter of the presentinvention, and (b) comparing the level of fluorescence resonance energytransfer (FRET) between the donor fluorescent moiety and the acceptorfluorescent moiety after contacting with that before contacting. Thelevel of FRET indicates the level of protein lysine2-hydroxyisobutyrylation in the sample. The level of FRET may beincreased or decreased after contacting.

A method for determining the level of proteinde-lysine-2-hydroxyisobutyrylation in a sample is also provided. Themethod comprises (a) contacting the sample with a fusion proteinreporter of the present invention, and (b) comparing the level offluorescence resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor fluorescent moiety after contactingwith that before contacting. The level of FRET indicates the level ofprotein de-lysine-2-hydroxyisobutyrylation in the sample. The level ofFRET may be increased or decreased after contacting.

For the determination method of the present invention, a sample may be abiological sample (e.g., bodily fluid or serum). The biological samplemay comprise a cell, a tissue biopsy, or a clinical fluid. Thebiological sample may be obtained from a subject (e.g., a mouse, rat, orhuman). The subject is healthy. The subject may have suffered from ormay be predisposed to a protein lysine 2-hydroxyisobutyrylation orde-lysine-2-hydroxyisobutyrylation related disorder, which may be anydisorder or disease linked to abnormal regulation of protein lysine2-hydroxyisobutyrylation or de-lysine-2-hydroxyisobutyrylation,respectively. Examples of such disorder or disease may include cancer,neurodegenerative diseases, aging, metabolic disorder, and dysgenesis.

The determination method of the present invention may further comprisecomparing the FRET level in the sample with a control FRET level. Thecontrol FRET level may be the FRET level in a control sample obtainedfrom a subject, who is healthy or has not suffered from or predisposedto a protein lysine 2-hydroxyisobutyrylation related disorder. The FRETlevel in the sample may be higher or lower than the control FRET level.

The determination method of the present invention may further compriseadding an agent to the sample. In some embodiments, the agent is knownto promote or inhibit protein lysine 2-hydroxyisobutyrylation. In otherembodiments, the agent is a screening candidate for a regulator ofprotein lysine 2-hydroxyisobutyrylation. The screening candidate may bea compound or a biological molecule.

For each determination method of the present invention, a kit isprovided. The kit comprises a fusion protein of the present invention.The kit may further comprise an instruction directing how to carry outthe method.

A kit for isolating a peptide containing a 2-hydroxyisobutyrylatedlysine is also provided. The kit comprises an isolated lysine2-hydroxyisobutyrylation specific affinity reagent capable of bindingspecifically to a peptide comprising a 2-hydroxyisobutyrylated lysine.

A method for treating or preventing a protein lysine2-hydroxyisobutyrylation related disease in a subject in need thereof isprovided. The method comprises administering to the subject an effectiveamount of a composition comprising an agent that regulates proteinlysine 2-hydroxyisobutyrylation. The agent may be a screen candidateidentified by a determination method of the present invention. Theprotein lysine-2-hydroxyisobutyrylation may be histonelysine-2-hydroxyisobutyrylation.

A method for treating or preventing a protein orde-lysine-2-hydroxyisobutyrylation related disease in a subject in needthereof is provided. The method comprises administering to the subjectan effective amount of a composition comprising an agent that regulatesprotein de-lysine-2-hydroxyisobutyrylation. The agent may be a screencandidate identified by a determination method of the present invention.The protein de-lysine-2-hydroxyisobutyrylation may be histonede-lysine-2-hydroxyisobutyrylation.

Example 1 Materials Cell Culture and Synchronization.

HeLa cells were cultured in DMEM containing 10% FBS, 1%penicillin/streptomycin at 37° C., 5% CO₂. HeLa cells were arrested atG2/M phase by a thymidine-nocodazole block. Briefly, at 50% confluency,HeLa cells were treated with 2 mM thymidine (Sigma, St Louis, Mo., USA)for 24 hours. After releasing cells from the thymidine block for 3hours, Nocodazole (Sigma, St Louis, Mo., USA) with a final concentrationof 100 ng/ml was added for another 12 hours. The dish was gently shakenand the medium containing the mitotic cells were collected. The cellswere washed with cold 1X PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10.0mmol/L Na₂HPO₄, 1.76 mmol/L NaH₂PO₄, pH=7.4) for 3 times and used forfurther downstream experiments.

Silac Labeling.

The SILAC media is formulated with SILAC Flex media supplemented with 10ml 200 g/liter glucose, 10 ml 200 mM L-glutamine, 1.5 ml 10 g/L PhenolRed solution, 100 ml Dialyzed FBS, 10 ml 100× penicillin-Streptomycin,and 100 mg/L ¹³C₆-L-lysine.HCl or 100 mg/liter non-labeled¹²C₆-L-lysine.HCl. The Final volume is 1 L after the above requiredcomponents have been added. All reagents were purchased from Invitrogen.The media were sterile filtered. Cells were washed with 1×PBS bufferprior to exposure to SILAC Flex media. Cells were passaged 1:3 throughdissociation in 0.25% trypsin. In the HeLa cells synchronizationexperiment, the thymine was dissolved in water without lysine andnocodazole was dissolved in DMSO without lysine. HeLa Cells underwentmany passages before synchronization to achieve high isotopic amino acidincorporation. The incorporation efficiency was determined by massspectrometry.

Chemical Propionylation.

Chemical propionylation before trypsin digestion procedure was showed asbelow: To 1.0 mg of histone sample dissolved in 300 μL of 0.1 M NH₄HCO₃buffer (pH=8.0), 5.0 μL propionic anhydride was added. The pH of mixturewas adjusted to keep pH between 8 and 9 by addition of 1 M NaOH withinstant monitor. When the pH didn't decrease to below 8, another 5.0 μLpropionic anhydride was added, and the pH of mixture was still kept pHbetween 8 and 9 by addition of 1 M NaOH until the pH didn't decrease.The propionylated histones were digested with trypsin (histone:trypsin=20:1) and used for immunoprecipitation. In chemicalpropionylation after trypsin digestion procedure, 20 μL instead of 5.0μL propionic anhydride was used in each step.

Immunoprecipitation.

Briefly, anti-K_(2ohibu) (PTM Biolabs, Inc. (Chicago, Ill.)),anti-K_(cr) or anti-K_(ac) antibodies was first immobilized to prewashedprotein A agarose beads (GE Healthcare Biosciences, Pittsburgh, Pa.) ata density of 5 mg of antibody per ml drained beads. The tryptic peptidesin NH₄HCO₃ solution were incubated with 15 μL antibody immobilizedprotein A beads at 4° C. overnight with gentle shaking. Afterincubation, the beads were washed three times with NETN buffer (50 mMTris.HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP40), twice with ETNbuffer (50 mM Tris.HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA) and once withwater. The bound peptides were eluted from the beads by washing threetimes with 30 μL of 0.1 M glycine solution (pH=2.5). The elutes werecombined and dried in a SpeedVac.

Results Identification of a Mass Shift of +86.0354 Da at a HistoneLysine Residue

In order to discover new histone PTMs, the histone proteins from mousespermatogenic cells, where a unique and genome-wide chromatin remodelingoccurs, were digested with trypsin and the resulting tryptic peptideswere analyzed by HPLC/MS/MS. The acquired MS/MS data was analyzed by thePTMap software, a non-restrictive sequence alignment algorithm thatenables to detect a mass shift caused by a PTM. The analysis identifieda modified H4 peptide, DAVTYTEHAKR (SEQ ID NO: 29), containing a massshift of +86.0354 Da at its lysine residue (or H4K77). The onlyreasonable elemental composition responsible for this mass shift wasC₄H₇O₂ (mass shift plus one proton) using ±0.02 Da mass tolerance and amaximum of 2 nitrogen atoms. According to the formula, we proposed fivepossible structures for the lysine modification: 2-hydroxyisobutyryl(K_(2ohibu)), 2-hydroxybutyryl (K_(2ohbu)), 3-hydroxybutyryl(K_(3ohbu)), 3-hydroxyisobutyryl (K_(3ohibu)) and 4-hydroxybutyryl(K_(4ohbu)) groups (FIG. 1).

The Mass Shift of +86.0354 Da is Caused by K_(2ohibu)

It is generally accepted that, if two peptides have the same MS/MSfragmentation patterns and are indistinguishable in HPLC chromatographicprofiles, they have identical structures. To determine which of the fivestructure isomers is responsible for the mass shift, we firstsynthesized five Fmoc-protected lysine derivatives each bearing one ofthe five possible modifications. We then used them for the synthesis offive peptides that have the same peptide sequence (DAVTYTEHAKR) (SEQ IDNO: 29), but the different hypothesized modifications added at lysineresidues, respectively.

We analyzed these synthetic peptides as well as the in vivo peptide byhigh-resolution MS. The MS/MS spectrum of the in vivo peptide (FIG. 2A)matched exactly with four of the synthetic peptides:DAVTYTEHAK_(2ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29) (FIG. 2A),DAVTYTEHAK_((±)-2ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29),DAVTYTEHAK_((r)-3ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29) andDAVTYTEHAK_((s)-3ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29). However, theMS/MS spectrum of the 4-hydroxybutyrylated peptide (DAVTYTEHAK_(4ohbu)R(DAVTYTEHAKR; SEQ ID NO: 29)) had a significant neutral loss peakresulting from a weight loss of 86.03 Da based on its calculatedmolecular weight, leading to a different spectrum from that of the invivo peptide. Thus, we concluded that the PTM in the in vivo peptide isnot lysine 4-hydroxybutyrylation.

Theoretically, two peptides with the same amino acid sequence andenantiomeric groups at the same PTM site will have the same retentiontimes on a nonchiral HPLC column. This conclusion was confirmed in ourco-elution experiment using the peptides DAVTYTEHAK_((r)-3ohbu)R(DAVTYTEHAKR; SEQ ID NO: 29) and DAVTYTEHAK_((s)-3ohbu)R (DAVTYTEHAKR;SEQ ID NO: 29). Therefore, in subsequent co-elution experiments, the useof racemic or enantiomeric peptides should have no influence on theco-elution results. To examine HPLC chromatographic profiles of the foursynthetic peptides, we mixed the four remaining synthetic peptides(DAVTYTEHAK_(2ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29),DAVTYTEHAK_((±)-2ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29),DAVTYTEHAK_((r)-3ohbu)R (DAVTYTEHAKR; SEQ ID NO: 29), andDAVTYTEHAK_((s)-3ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29)) together with thein vivo one. The HPLC/MS/MS results revealed that these four peptideshad different retention times and could be distinguished from the invivo peptide bearing a mass shift of +86.0354 Da in the HPLC system(FIG. 2B). Accordingly, the pair-wise co-elution experiments can be usedto determine the four putative structures.

Among the four possible structures, we found that the in vivo peptideco-eluted perfectly with the synthetic 2-hydroxyisobutyrylated peptideDAVTYTEHAK_(2ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29) (FIG. 2C). However,the other three synthetic peptides, DAVTYTEHAK_((±)-2ohbu)R(DAVTYTEHAKR; SEQ ID NO: 29), DAVTYTEHAK_((r)-3ohbu)R (DAVTYTEHAKR; SEQID NO: 29), and DAVTYTEHAK_((s)-3ohibu)R (DAVTYTEHAKR; SEQ ID NO: 29),did not co-elute with the in vivo peptide. These results indicate thatthe mass shift of the in vivo peptide is caused by K_(2ohibu) instead oflysine 2-hydroxybutyrylation, 3-hydroxybutyrylation, and3-hydroxyisobutyrylation. Together, the MS/MS and HPLC co-elutionexperiments demonstrated that the mass shift of +86.0354 Da at thelysine residue of the peptide DAVTYTEHAKR (SEQ ID NO: 29) is caused byK_(2ohibu).

To further confirm the new modification K_(2ohibu), we examined anadditional peptide, VTIMPK_(+86.03)DIQLAR (SEQ ID NO: 47) with a massshift of +86.03 Da, which were derived from mouse spermatogenic cellshistones H3. These experiments again unambiguously demonstrated that themass shift of +86.03 Da was caused by K_(2ohibu). Taken together, wetested the five possible structures of a lysine PTM caused by a massshift of +86.03 Da in three in vivo peptides by MS/MS and HPLCco-elution experiments. Our results clearly showed that the mass shiftof +86.03 Da at histone lysine residues is caused by K_(2ohibu).

Verification of Histone K_(2ohibu) by Western Blotting

To corroborate the new PTM, we generated a pan anti-K_(2ohibu) antibodyand a sequence-specific antibody against H4K8_(2ohibu). Both antibodieshave good specificities based on dot-spot assays. Using Western blot, wespecifically detected K_(2ohibu) signal among all the HeLa cells corehistone proteins. Likewise, using anti-H4K8_(2ohibu) antibody, wedetected H4K8_(2ohibu) in HeLa cell histones, but not recombinant H4(FIG. 3A). These results clearly demonstrate the existence of K_(2ohibu)on human histones.

Histone K_(2ohibu) is an Evolutionarily Conserved Mark

Using the pan anti-K_(2ohibu) and sequence-specific anti-H4K8_(2ohibu)antibodies, we detected K_(2ohibu) on histones not only from HeLa cells,but also from mouse embryonic fibroblast (MEF) cells, Drosophila S2cells and yeast S. cerevisiae cells (FIG. 3B). This result clearlyindicates that K_(2ohibu) is an evolutionarily-conserved in eukaryoticcells. To identify histone K_(2ohibu) sites, we used anaffinity-directed MS method, involving tryptic digestion of theextracted histones from the cells of interest, enrichment of K_(2ohibu)peptides with anti-K_(2ohibu) antibody, HPLC/MS/MS analysis and proteinsequence database search. This study led to identification of 60 histoneK_(2ohibu) sites in mouse male germ cells (Table 1) and 22 histoneK_(2ohibu) sites in HeLa cells (Table 2).

Several interesting features were observed among histone K_(2ohibu)sites. First, we detected 63 K_(2ohibu) sites in human and mousehistones. For 27 of these 63 sites, neither acetylation norcrotonylation had been reported by the research community (FIG. 3D).Second, K_(2ohibu) is located not only in N-terminal domains but also inother regions of core histones, a profile different from K_(ac). As anexample, eight K_(2ohibu) sites were found to be located in the H2Bglobular domain between lysine 34 to lysine 120. We further examined thelocations of K_(2ohibu) lysine residues in the H2B core region and foundthat they are precisely located at surfaces involved in inter- orintra-nucleosome interactions in mouse spermatogenic cells (FIG. 3C).For example, H2BK43 in the L1 loop, H2BK85 in the L2 loop and H2BK34 arein direct contact with DNA. Accordingly, a change of charge state frompositive (protonated amine group) to neutralized state(2-hydroxyisobutyrylated lysine) is highly likely to impact theassociation of H2B with DNA. These observations suggest different rolesof histone K_(2ohibu) from histone K_(ac) and K_(cr) in the regulationof chromatin structure and function. More interestingly, the occurrenceof these nucleosome-destabilizing PTMs in the mouse spermatogenic cellsis indicative of their potential role in the large-scale nucleosomedisassembly that takes place in the post-meiotic cells.

To determine the stoichiometry of histone K_(2ohibu) marks, we analyzedtryptic digests of core histones from unsynchronized and synchronizedHeLa cells using a Stable isotope labeling by amino acids in cellculture (SILAC)-based method. Table 5 shows the absolute stoichiometryanalysis of K_(2ohibu) sites during mitosis. The stoichiometries ofhistone K_(2ohibu) sites were calculated based on a previously publishedmethod which requires the quantification ratios of proteins, K_(2ohibu)peptides and the corresponding unmodified tryptic peptides. BothK_(2ohibu) peptides and protein quantification ratios were measured byMaxquant (v1.0.13.13) and the protein ratios were calculated using onlyunmodified peptides. The quantification ratios of the correspondingunmodified tryptic peptides were based on the Maxquant quantificationratios of the longest completely-cleaved tryptic form ofK_(2ohibu)-bearing peptides. We were able to determine stoichiometry offour K_(2ohibu) sites, H3K79, H2BK108, H4K91 and H1.2K63. Theirstoichiometry reaches to 5.33% and 7.79% for H4K91 and H1.2K63,respectively, in the cells that were synchronized to G2/M phase.Stoichiometries for many K_(ac) and methylation sites are lower than afew percentages. As an example, it was reported that the abundance ofhistone H3 lysine 56 acetylation (H3K56_(ac)), a histone mark with arole in genomic stability, is less than 0.1%. Our results suggest thatthe abundance of histone K_(2ohibu) marks is likely to be in line withthat of histone acetylation and methylation marks with low to mediumabundance.

Labeling of Histone K_(2ohibu) Marks by Isotopic 2-Hydroxyisobutyrate

The short-chain acyl-CoAs are the donor molecules for lysine acylations,e.g., acetyl-CoA for K_(ac) reaction. Thus, most likely,2-hydroxyisobutyryl-CoA is the cofactor for K_(2ohibu) reaction.2-Hydroxyisobutyryl-CoA is an intermediate in bacteria for theproduction of 2-hydroxyisobutyric acid, a building block for industrialpolymer synthesis, and for the degradation of fuel oxygenates, methyland ethyl tert-butyl ether, in bacteria and yeast. In human,2-hydroxyisobutyric acid is one of the detectable organic acids that areassociated with lactic acidosis.

To test if 2-hydroxyisobutyrate could be used by cells for K_(2ohibu)via 2-hydroxyisobutyryl-CoA in a similar way as cells use acetate forlysine acetylation, we treated HeLa cells with 50 mMD₆-2-hydroxyisobutyrate for 72 hours. The isotopically labeled histoneK_(2ohibu) peptides were analyzed using MS. Our analysis detectedD₆-labeled histone peptides. Table 6 shows the mass spectrometricanalysis of the isotopic HeLa cells histone sample. The HeLa cells weretreated with 50 mM D₆₋₂-hydroxyisobutyrate for 72 hours in DMEM medium.Only D₆-labeled K_(2ohibu) sites were shown. This result implies that2-hydroxyisobutyryl-CoA, originated from the precursor2-hydroxyisobutyrate, is likely the cofactor for K_(2ohibu).

Spatio-Temporal Labeling of Histone H4K8 by 2-Hydroxyisobutyrylation andAcetylation in Spermatogenic Cells

We then examined the roles of H4K8_(2ohibu) in the male germ cellspecific transcriptional activities and chromatin remodeling. Indeed,our previous investigations of a critical spermatogenic factor, Brdt,specifically recognizing histone H4 lysine 5 acetylation (H4K5_(ac)) andH4K8_(ac), indicate the important function of these two H4 lysineresidues, in the control of male germ cell gene expression.

Toward this goal, we first analyzed spermatogenic cells byimmunohistochemistry (IH) to examine the global dynamics ofH4K8_(2ohibu). The genome-wide distribution of this histone mark isdynamic and varies as a function of male germ cell differentiation (FIG.4A). A clear labeling is observed in spermatogonia, which decreases inmeiotic cells. In spermatocytes, the H4K8_(2ohibu) labeling does notshow any particular pattern, while in round spermatids, H4K8_(2ohibu)forms a dot-like nuclear structure. At later stages, in elongatingspermatids undergoing histone-to-transition protein (TP) replacement,the labeling becomes intense and genome-wide (FIG. 4A). Remarkably, aparallel analysis of H4K8_(ac), a mark previously studied inspermatogenic cells, revealed a similar pattern of staining asH4K8_(2ohibu) (FIG. 4B). Interestingly, the specific pattern ofH4K8_(ac) labeling in round spermatids was also observed.

In order to understand the nature of the dot-like structure observed inround spermatids on the histological testis sections, we investigatedthe dynamics of H4K8_(2ohibu) using fluorescent immunodetection. In themouse round spermatids, both H4K8_(2ohibu) and H4K8_(ac) marked a uniquenuclear structure. In addition, the chromocenter, formed by theaggregation of all centromeric and pericentric chromosomal domains,forms a unique large heterochromatic structure, which is clearly visibleby DAPI staining. The analysis of this structure with H4K8_(2ohibu)staining shows the absence of this mark from the chromocenter, but theantibody labels an adjacent structure (FIG. 4C). The co-detection ofHP1gamma, known to mark both the chromocenter and the adjacent sexchromosomes, together with H4K8_(2ohibu), demonstrated that theH4K8_(2ohibu)-enriched structure corresponds to the sex chromosome,since it is absent from the chromocenter and perfectly co-localizes withthe sex chromosomes. Interestingly, here again, the H4K8_(ac) mark showsa similar pattern as H4K8_(2ohibu), which is consistent with previousobservations on the preferential association of this mark with sexchromosomes.

High-Resolution Genome-Wide Mapping of H4K8_(2ohibu) and H4K8_(ac) inMeiotic and Post-Meiotic Cells

To gain functional insights into histone H4K8_(2ohibu) and H4K8_(ac), weprepared nuclei from enriched meiotic (spermatocytes) and post-meiotic(round spermatids) cells and used them for a high-resolution mapping ofthese marks in genomic localization by ChIP-seq. Our study shows thatboth marks are dynamic and that there is a significant re-distributionof H4K8_(2ohibu) peaks between spermatocytes and round spermatids (FIG.5A, upper). In addition, more than half of H4K8_(ac) peaks areassociated with nucleosomes bearing H4K8_(2ohibu) mark (FIG. 5A,bottom).

The sex chromosomes (mainly the X) have a distinct global peaksdistribution, where both marks are depleted compared to the autosomes,especially in spermatocytes (FIGS. 5C and 5D). This observationsuggested that H4K8_(2ohibu), like H4K8_(ac), could be associated withthe transcriptional activity of the chromosomes and that the relativedepletion of these marks, on the X chromosome in spermatocytes, could belinked to the sex chromosome inactivation in these cells. Interestingly,in agreement with the immunodetection data, an increase of H4K8_(2ohibu)peaks was observed on the X chromosome after the completion of meiosis(FIG. 5D). A similar phenomenon was observed for H4K8_(ac), althoughwith a much lesser degree than H4K8_(2ohibu) (FIG. 5C).

Depletion of H4K8_(ac) in the sex chromosomes in meiotic andpost-meiotic cells is generally associated with the transcriptionalsilencing of the chromosomes. The similar observation for the depletionof H4K8_(2ohibu) in spermatogenic cells suggests an association of thishistone mark with gene expression. Indeed, a metagene analysis supportedthis hypothesis, showing that H4K8_(2ohibu) and H4K8_(ac) are bothenriched at the transcriptional start sites (TSSs) of genes (FIG. 5B).Therefore, this result highlights H4K8_(2ohibu)'s possible roles intranscriptional control.

Epigenetic Gene Signposting by H4K8_(2ohibu) and H4K8_(ac) in Meioticand Post-Meiotic Cells

These data prompted us to examine the gene-containing fractions of thegenome that are associated with these two marks. H4K8_(2ohibu) peakswere found associated to 8855 genes in meiotic or post-meioticspermatogenic cells (FIG. 6A). In both spermatocytes and in roundspermatids, only a minor fraction of genes was associated withnucleosomes bearing H4K8_(ac) alone, while the vast majority of theH4K8_(ac)-associated genes (93%) were co-localized in the genome withH4K8_(2ohibu) (FIG. 6A). The gene expression patterns of the genesmarked by H4K8_(2ohibu), either alone or with H4K8_(ac) (43% and 57% ofH4K8_(2ohibu)-associated genes, respectively) were analyzed usingtranscriptomic data derived from normal mouse tissues (data available onthe GEO website, GSE10744, GSE9954, GSE4193 and GSE21749), as previouslydescribed. Briefly, within the list of genes found associated withH4K8_(2ohibu), we looked for those preferentially expressed in testiscompared to other tissues. This study showed that 29% theH4K8_(2ohibu)-associated genes are testis-specific, and include meioticand post-meiotic genes (FIGS. 6B and 6C).

Next, we examined if the genes associated with these two marks, alone orin combination, would show different transcriptional activities. To doso, stage-specific spermatogenic transcriptomes were downloaded (fromthe same studies as described above) and the mean expression levels ofthe gene categories associated with one or two marks were calculated. Inspermatocytes, the expression levels of the genes bearing H4K8_(2ohibu)alone are higher than those bearing none of the two H4K8 histone marks(FIG. 6D). In addition, combination of both marks slightly increases theeffect. Unexpectedly, in round spermatids, H4K8_(ac)-marked genes do notshow a different transcriptional activity compared to genes devoid ofthe two marks, which is different from our observations in spermatocytes(FIG. 6D, right). Interestingly, in these cells, the association ofH4K8_(2ohibu) alone with genes appeared as a strong indicator of ahigher transcriptional activity and a combination of both marks furtherenhance gene expression.

Because of the relative enrichment of H4K8_(2ohibu) in the post-meioticsex chromosomes observed by both ChIP-seq and immunodetection, wewondered if there could be a relationship between H4K8_(2ohibu) andhistone crotonylation, a histone mark that has previously been shownincreased on the sex chromosomes in post-meiotic cells. Interestingly,nearly no sex-linked gene was found associated with H4K8_(ac) alone(FIG. 6E, right panel), despite a moderate enrichment of this mark inpost-meiotic sex chromosomes and its apparent concentration on the sexchromosomes by immunodetection observed by us here and by others.Moreover, a relatively low proportion of sex chromosome-linked genes(18%) were associated with H4K8_(2ohibu), while, in contrast, this markwas present on half of the autosomal genes (FIG. 6E, left panel).Interestingly, nearly all sex chromosome-linked genes associated withH4K8_(2ohibu) were also included in the list of genes associated withhistone crotonylation, where a pan—instead of sequence-specificanti-H4K8_(cr) antibody was used. Taking into account the fact that nohistone acetylation was found associated with these genes, thisobservation suggests that histone K_(2ohibu), could be actually moredirectly involved than acetylation in activating the expression of atleast a fraction of these genes in round spermatids, in the repressivecontext of the sex chromosomes.

Discussion

In this study, we identified the in vivo histone K_(2ohibu) as a newhistone mark. This PTM was robustly validated by (i) MS/MS analysis,(ii) HPLC co-elution, and (iii) Western blotting using pan andsequence-specific anti-K_(2ohibu) antibodies. We subsequently identified63 K_(2ohibu) sites in histones, including 60 sites in mousespermatogenic cells and 22 sites in human HeLa cells, more than thetotal number of histone K_(ac) sites. Therefore, these histoneK_(2ohibu) sites add new elements to “histone language” andsignificantly enhance its complexity.

Diverse differences were observed among K_(2ohibu) K_(ac) and K_(cr).First, the three histone marks show different patterns in differentspermatogenic stages. These differences were consistently observed usingChIP-seq experiment. The immunostaining demonstrated that the histoneH4K8_(2ohibu) mark does not show any particular pattern inspermatocytes, but forms a dot-like nuclear structure in roundspermatids cells, before becoming genome-wide at the time of histonereplacement. Thus, K_(2ohibu) is not only functionally distinct fromK_(ac), but also has regulatory mechanisms different from those ofK_(ac).

Second, K_(2ohibu), K_(ac) and K_(cr) are located at different residuesof histones (FIG. 3C). Our study identified 63 histone K_(2ohibu) marks,more than the number of known histone K_(ac) marks. The K_(2ohibu) notonly exists at the N-termini of the histones, but also at their mainglobular domains, while the majority of known K_(cr) and K_(ac) occur atthe N-termini of the histones. Importantly, the stoichiometry of fourK_(2ohibu) sites (H3K79, H2BK108, H4K91 and H1.2K63) is comparable oreven higher than that of many histone K_(ac) marks with known biologicalfunctions.

Third, K_(ac) and K_(2ohibu) mark different groups of genes inspermatocytes. Interestingly, our study of genes associated withH4K8_(2ohibu) show that this mark is a better indicator of theirtranscriptional activity in post-meiotic round spermatids than theacetylation at the same position.

Fourth, K_(2ohibu) is structurally very different from lysinemethylation, K_(ac), or K_(cr). Table 7 shows a comparison of changes ofcharge status, size, hydrophobicity caused by lysine, lysinedimethylation, K_(ac), K_(2ohibu) and K_(cr). The hydrophobicity isdetermined based on calculated Log P value using Chemdraw software, inwhich the protonated residues were used for unmodified lysine anddimethylated lysine residues. “+” means a tendency to increase. “−”means a tendency to decrease. K_(2ohibu) not only neutralizes thepositive charge of lysine, but also induces much large change of itssize. More importantly, K_(2ohibu) has a hydroxyl group that enables themodified lysine to form hydrogen bonds with other molecules. Such ahydroxyl group is known to be important for the regulation of proteinfunctions, e.g., HIF1.

Finally, K_(2ohibu) is likely to result from the use of2-hydroxyisobutyryl-CoA. Cellular metabolism has been suggested to beclosely linked with epigenetic mechanisms. Thus, the K_(2ohibu) pathwaycould provide an opportunity for cells to reprogram epigenetics networksthrough histone modifications, in response to the dynamic change of acellular metabolite, 2-hydroxyisobutyryl-CoA.

A detailed analysis of H4K8_(2ohibu) in parallel with H4K8_(ac) allowedus to highlight a new epigenetic determinant of transcriptionalactivation. Indeed, when we consider the gene fraction of our ChIP-seqdata, we found that H4K8_(2ohibu) is a major mark associated with genetranscriptional activity and that H4K8_(ac)-bearing genes are largelyincluded in this category constituting a sub-population of theH4K_(2ohibu)-labelled genes. Interestingly, in post-meiotic cells,H4K8_(2ohibu), but not H4K8_(ac) alone, indicates gene activity and theaddition of H4K8_(ac) is associated to a group of genes with higheractivity. These observations suggest that the majortranscription-associated mark in the post-meiotic spermatogenic cells isunexpectedly not H4K8_(ac) but H4K8_(2ohibu). This hypothesis receivesstrong support from a precise analysis of the situation of the sexchromosomes. Indeed, the chromosome-wide transcriptional inactivationthat occurs in meiotic cells is found here associated with a depletionof both marks, in agreement with their involvement in gene activity.

However, we also show here that in post-meiotic round spermatids,H4K8_(2ohibu), but not H4K8_(cr), becomes associated with a faction ofX-linked genes that is also labeled with histone crotonylation. Theremight be in fact a functional redundancy between H4K8_(2ohibu) andK_(cr) in this process. However, since pan anti-K_(cr) antibody was usedin the previous ChIP-seq study, some specific histone K_(cr) sites otherthan H4K8, may mark similar genomic locations as histone H4K8_(2ohibu).Based on these observations, we propose that H4K_(2ohibu), althoughfunctionally similar to H4K8_(ac) in terms of transcriptionalactivation, is of much wider use and ensures unique functions when geneactivation needs to take place under general repressive conditions.

Another interesting observation is the disparity between ChIP-seq dataand immunostaining data obtained in the post-meiotic sex chromosomeswith anti-H4K8_(2ohibu) and H4K8_(ac) antibodies. Indeed, intenselabeling of the sex chromosomes with the two histone marks was detectedon testis sections by IH or by IF on seminiferous tubule sections.However, ChIP-seq data show that, although these marks are relativelyenriched on the post-meiotic compared to the meiotic sex chromosomes,they remain relatively depleted on these chromosomes compared to theautosomes. Interestingly, the apparent enrichment of H4K8_(ac) in thepost-meiotic sex chromosomes observed by in situ staining has beenpreviously described. This may be caused by a specific 3D organizationof the sex chromosomes in round spermatids, which would create a localregion with a high density of histone marks or with chromatinorganization-dependent histone marks exposed for antibody recognition.We had previously described a similar phenomenon regarding the macroH2Ahistone variant. While the inactive X chromosome was intensely-labeledwith anti-macroH2A antibodies, the enrichment of this histone variant onthe X chromosome-linked genes was only moderate compared to autosomalgenes.

The discovery of K_(2ohibu) is just the beginning of the journey tostudy the possible diverse functions of this new modification. Extensivestudy of histone K_(ac) and methylation in the past few decades hasrevealed their critical roles in epigenetics and transcriptionalcontrol. A similar biological potential is likely to exist for histoneK_(2ohibu). In addition, given the fact that all the known histone PTMsare also present in non-histone proteins, it is anticipated thatK_(2ohibu) should be present in non-histone proteins and should havenucleosome-independent functions as well. Future identification of theregulatory enzymes for K_(2ohibu) and of its non-histone substrates willaccelerate the characterization of its chromosome-independent functions.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a percentage, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably ±l%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts, and other references cited herein areincorporated by reference in their entirety. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

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TABLE 1 K_(2ohibu) sites in human histone proteins Human SEQ HistoneK_(2ohibu) ID Protein sites Peptide sequences NO H1.2 H1.2K45ASGPPVSELITK_(2ohibu)AVAASK 30 H1.2 H1.2K51 AVAASK_(2ohibu)ER 31 H1.2H1.2K62 SGVSLAALK_(2ohibu)K 32 H1.2 H1.2K63 K_(2ohibu)ALAAAGYDVEK 33H1.2 H1.2K84 LGLK_(2ohibu)SLVSK 34 H1.2 H1.2K89 SLVSK_(2ohibu)GTLVQTK 35H1.2 H1.2K96 GTLVQTK_(2ohibu)GTGASGSFK 36 H2A H2AK95NDEELNK_(2ohibu)LLGK 37 H2B H2BK5 PEPSK_(2ohibu)SAPAPK 38 H2B H2BK46VLK_(2ohibu)QVHPDTGISSK 39 H2B H2BK85 LAHYNK_(2ohibu)R 40 H2B H2BK108LLLPGELAK_(2ohibu)HAVSEGTK 41 H2B H2BK116 HAVSEGTK_(2ohibu)AVTK 42 H2BH2BK120 AVTK_(2ohibu)YTSSK 43 H3 H3K23 QLATK_(2ohibu)AAR 44 H3 H3K56YQK_(2ohibu)STELLIR 45 H3 H3K79 EIAQDFK_(2ohibu)TDLR 46 H3 H3K122VTIMPK_(2ohibu)DIQLAR 47 H4 H4K31 DNIQGITK_(2ohibu)PAIR 48 H4 H4K77DAVTYTEHAK_(2ohibu)R 29 H4 H4K79 _(2ohibu)KTVTAMDVVYALK 49 H4 H4K91TVTAMDVVYALK_(2ohibu)R 50

TABLE 2 K_(2ohibu) sites in mouse histone proteins Mouse SEQ HistoneK_(2ohibu) ID Protein sites Peptide sequences NO H1.2 H1.2K22K_(prop)K_(2ohibu)AAK_(prop)K_(prop)PAGVR 51 H1.2 H1.2K25K_(prop)K_(prop)AAK_(2ohibu)K_(prop)PAGVR 51 H1.2 H1.2K26K_(prop)AAK_(prop)K_(2ohibu)PAGVR 52 H1.2 H1.2K33K_(2ohibu)ASGPPVSELITK_(prop)AVAASK_(2ohibu) 53 H1.2 H1.2K51K_(prop)ASGPPVSELITK_(prop)AVAASK_(2ohibu)ER 54 H1.2 H1.2K62SGVSLAALK_(2ohibu)K 32 H1.2 H1.2K74 ALAAAGYDVEK_(2ohibu)NNSR 55 H1.2H1.2K80 IK_(2ohibu)LGLK 56 H1.2 H1.2K84 _(prop)LGLK_(2ohibu)SLVSK_(prop)34 H1.2 H1.2K96 _(prop)GILVQTK_(2ohibu)GTGASGSFK_(prop) 57 H1.2 H1.2K109_(prop)K_(2ohibu)AASGEAK_(prop)PQAK_(prop) 58 H1.2 H1.2K116_(prop)AASGEAK_(2ohibu)PQAK_(prop) 59 H1.2 H1.2K120_(prop)AASGEAK_(prop)PQAK_(2ohibu)K_(prop) 60 H1.2 H1.2K128_(prop)AK_(2ohibu)K_(prop)PAGAAK_(prop) 61 H1.2 H1.2K135_(prop)AK_(prop)K_(prop)PAGAAK_(2ohibu)K_(prop)PK_(prop) 62 H1.2H1.2K147 _(prop)K_(prop)ATGAATPK_(2ohibu)K_(prop) 63 H1.2 H1.2K158_(prop)K_(prop)AK_(2ohibu)K_(prop)PAAAAVTK_(prop) 64 H1.2 H1.2K167_(prop)K_(prop)PAAAAVTK_(2ohibu)K_(prop) 65 H1.2 H1.2K211K_(prop)VAAK_(prop)K_(prop)K_(2ohibu) 66 H2A H1.2AK5,GK_(2ohibu)QGGK_(2ohibu)AR 67 K9 H2A H2AK36 K_(2ohibu)GNYSER 68 H2AH2AK74, DNK_(2ohibu)K_(2ohibu)TR 69 K75 H2A H2AK95 NDEELNK_(2ohibu)LLGR70 H2A H2AK118 VTIAQGGVLPNIQAVLLPK_(2ohibu)K 71 H2B H2BK5_(prop)PEPAK_(2ohibu)SAPAPK_(prop) 72 H2B H2BK12_(prop)PEPAK_(prop)SAPAPK_(prop)K_(2ohibu)GSK 73 H2B H2BK20K_(prop)AISK_(2ohibu)AQK_(prop) 74 H2B H2BK23AVTK_(prop)AQK_(2ohibu)K_(prop)DGK_(prop)K_(prop)R 75 H2B H2BK24AVTK_(prop)AQK_(prop)K_(2ohibu)DGK_(prop)K_(prop)R 75 H2B H2BK34K_(2ohibu)ESYSVYVYK 76 H2B H2BK43 KESYSVYVYK_(2ohibu)VLK 77 H2B H2BK46VLK_(2ohibu)QVHPDTGISSK 39 H2B H2BK57QVHPDTGISSK_(2ohibu)AMGIMNSFVNDIFER 78 H2B H2BK85 LAHYNK_(2ohibu)R 40H2B H2BK108 LLLPGELAK_(2ohibu)HAVSEGTK 41 H2B H2BK116HAVSEGTK_(2ohibu)AVTK 42 H2B H2BK120 AVTK_(2ohibu)YTSSK 43 H3 H3K4TK_(2ohibu)QTAR 79 H3 H3K9 K_(2ohibu)STGGK_(ac)APR 80 H3 H3K14K_(prop)STGGK_(2ohibu)APR 80 H3 H3K18 K_(2ohibu)QLATK_(ac)AAR 81 H3H3K23 KQLATK_(2ohibu)AAR 81 H3 H3K27K_(2ohibu)SAPATGGVK_(prop)K_(prop)PHR 82 H3 H3K36K_(prop)SAPATGGVK_(2ohibu)K_(prop)PHR 82 H3 H3K56 YQK_(2ohibu)STELLIR 45H3 H3K64 K_(2ohibu)LPFQR 83 H3 H3K79 EIAQDFK_(2ohibu)TDLR 46 H3 H3K122VTIMPK_(2ohibu)DIQLAR 47 H4 H4K5 K_(2ohibu)GGK_(ac)GLGK_(ac)GGAK_(ac)R84 H4 H4K8 GK_(ac)GGK_(2ohibu)GLGK_(ac)GGAK_(ac)R 85 H4 H4K12GLGK_(2ohibu)GGAK_(ac)R 86 H4 H4K16 GGK_(prop)GLGK_(prop)GGAK_(2ohibu)R87 H4 H4K31 DNIQGITK_(2ohibu)PAIR 48 H4 H4K44 RGGVK_(2ohibu)R 88 H4H4K59 GVLK_(2ohibu)VFLENVIR 89 H4 H4K77 DAVTYTEHAK_(2ohibu)R 29 H4 H4K79K_(2ohibu)TVTAMDVVYALK 49 H4 H4K91 KTVTAMDVVYALK_(2ohibu)R 90

TABLE 3 K_(2ohibu) sites in S. cerevisiae histone proteins S. cerevisiaeSEQ Histone K_(2ohibu) ID Protein sites Peptide sequences NO H2A H2AK13AGSAAK_(2ohibu)ASQSR 91 H2B H2BK37 K_(2ohibu)ETYSSYIYK 92 H2B H2BK46ETYSSYIYK_(2ohibu)VLK 93 H2B H2BK82 IATEASK_(2ohibu)LAAYNK 94 H2BH2BK111 LILPGELAK_(2ohibu)HAVSEGTR 95 H3 H3K56 FQK_(2ohibu)STELLIR 96 H4H4K31 DNIQGITK_(2ohibu)PAIR 48 H4 H4K77 DSVTYTEHAK_(2ohibu)R 97 H4 H4K79K_(2ohibu)TVTSLDVVYALK 98 H4 H4K91 TVTSLDVVYALK_(2ohibu)R 99

TABLE 4 K_(2ohibu) sites in Tetrahymena macronuclear  histone proteinsTetrahymena SEQ Histone K_(2ohibu) ID Protein sites Peptide sequences NOH4 H4K11 GMGK_(2ohibu)VGAK 100 H4 H4K79 K_(2ohibu)TVTAMDVVYALK  49 H4H4K91 TVTAMDVVYALK_(2ohibu)R  50 H3 H3K27 K_(2ohibu)SAPATGGIK 101 H3H3K56 YQK_(2ohibu)STDLLIR 102 H3 H3K64 K_(2ohibu)LPFQR  83 H3 H3K122VTIMTK_(2ohibu)DMQLAR 103 H2B H2BK4 K_(2ohibu)APAAAAEK 104 H2B H2BK12K_(ac)APAAAAEK_(2ohibu)K 105 H2B H2BK41 VLK_(2ohibu)QVHPDVGISK 106 H2BH2BK74 IALESS_(2ohibu)LVR 107 H2B H2BK111 HAISEGTK_(2ohibu)AVTK 108 H2BH2BK115 AVTK_(2ohibu)FSSSTN 109 H2A H2AK17 TASSK_(2ohibu)QVSR 110

TABLE 5Absolute stoichiometry analysis of K_(2ohibu) sites during mitosis.Stoichiometry Stoichiometry  of K_(2ohibu) in of K_(2ohibu) inK_(2ohibu) unsynchronized G2/M phase HeLa sites Peptide sequencesHeLa cells cells H3K79 EIAQDFK_(2ohibu)TDLR 0.60% 1.45% H2BK108LLLPGELAK_(2ohibu)HAVSEGTK 0.64% 1.54% H4K91 TVTAMDVVYALK_(2ohibu)R3.16% 5.33% H1.2K63 SGVSLAALK_(2ohibu)K 3.34% 7.79%

TABLE 6 Mass spectrometric analysis of the isotopic HeLacells histone sample. D₆-labeled K_(2ohibu) sites Peptide sequencesSEQ ID NO H2AK7 AGGK_(ac)AGK_(2ohibu)DSGK 111 H2AK4AGGK_(2ohibu)AGK_(ac)DSGK 111 H4K8 GGK_(2ohibu)GLGK_(ac)GGAK_(ac)R  87H4K12 GGK_(ac)GLGK_(2ohibu)GGAK_(ac)R  87 H4K12 GLGK_(2ohibu)GGAK_(ac)R 86 H3K23 K_(ac)QLATK_(2ohibu)AAR  81 H2BK5 PEPAK_(2ohibu)SAPAPK  72H2BK11 SAPAPK_(2ohibu)K_(ac)GSK 112

TABLE 7 A comparison of changes of charge status, size, hydrophobicitycaused by lysine, lysine dimethylation, K_(ac), K_(2ohibu) and K_(cr).PTMs                       comparison          

       

   

   

pl 0 + − − − Size 0 + + +++ ++ Hydrophobicity 0 +++ + ++ ++++

What is claimed:
 1. A method for producing an isolated affinity reagentthat binds specifically to a protein or a fragment thereof comprising alysine when the lysine is 2-hydroxyisobutyrylated, wherein the isolatedaffinity reagent does not bind to the protein or fragment thereof whenthe lysine is not 2-hydroxyisobutyrylated, comprising immunizing a hostwith a peptide comprising a 2-hydroxyisobutyrylated lysine.
 2. Themethod of claim 1, wherein the binding of the isolated affinity reagentto the protein or a fragment thereof depends on the2-hydroxyisobutyrylated lysine and a surrounding peptide sequencethereof, and wherein the surrounding peptide sequence comprises at leasttwo residues on each of the N-terminal and C-terminal sides of the2-hydroxyisobutyrylated lysine.
 3. The method of claim 1, wherein thepeptide further comprises at least two amino acid residues on each ofthe N-terminal and C-terminal sides of the 2-hydroxyisobutyrylatedlysine.
 4. The method of claim 1, wherein the peptide comprises an aminoacid sequence at least 50% identical to a sequence selected from thegroup consisting of DAVTYTEHAK2ohibuR (SEQ ID NO: 29),ASGPPVSELITK2ohibuAVAAS (SEQ ID NO: 30), AVAASK2ohibuER (SEQ ID NO: 31),SGVSLAALK2ohibuK (SEQ ID NO: 32), K2ohibuALAAAGYDVEK (SEQ ID NO: 33),LGLK2ohibuSLVSK (SEQ ID NO: 34), propLGLK2ohibuSLVSKprop (SEQ ID NO:34), SLVSK2ohibuGTLVQTK (SEQ ID NO: 35), GTLVQTK2ohibuGTGASGSFK (SEQ IDNO: 36), NDEELNK2ohibuLLGK (SEQ ID NO: 37), PEPSK2ohibuSAPAPK (SEQ IDNO: 38), VLK2ohibuQVHPDTGISSK (SEQ ID NO: 39), LAHYNK2ohibuR (SEQ ID NO:40), LLLPGELAK2ohibuHAVSEGTK (SEQ ID NO: 41), HAVSEGTK2ohibuAVTK (SEQ IDNO: 42), AVTK2ohibuYTSSK (SEQ ID NO: 43), QLATK2ohibuAAR (SEQ ID NO:44), YQK2ohibuSTELLIR (SEQ ID NO: 45), EIAQDFK2ohibuTDLR (SEQ ID NO:46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47), DNIQGITK2ohibuPAIR (SEQ ID NO:48), K2ohibu TVTAMDVVYALK (SEQ ID NO: 49), TVTAMDVVYALK2ohibuR (SEQ IDNO: 50), KpropK2ohibuAAKpropKpropPAGVR (SEQ ID NO: 51),KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51), KpropAAKpropK2ohibuPAGVR(SEQ ID NO: 52), K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 5. The method of claim 1, wherein the peptidecomprises an amino acid sequence selected from the group consisting ofDAVTYTEHAK2ohibuR (SEQ ID NO: 29), ASGPPVSELITK2ohibuAVAAS (SEQ ID NO:30), AVAASK2ohibuER (SEQ ID NO: 31), SGVSLAALK2ohibuK (SEQ ID NO: 32),K2ohibuALAAAGYDVEK (SEQ ID NO: 33), LGLK2ohibuSLVSK (SEQ ID NO: 34),propLGLK2ohibuSLVSKprop (SEQ ID NO: 34), SLVSK2ohibuGTLVQTK (SEQ ID NO:35), GTLVQTK2ohibuGTGASGSFK (SEQ ID NO: 36), NDEELNK2ohibuLLGK (SEQ IDNO: 37), PEPSK2ohibuSAPAPK (SEQ ID NO: 38), VLK2ohibuQVHPDTGISSK (SEQ IDNO: 39), LAHYNK2ohibuR (SEQ ID NO: 40), LLLPGELAK2ohibuHAVSEGTK (SEQ IDNO: 41), HAVSEGTK2ohibuAVTK (SEQ ID NO: 42), AVTK2ohibuYTSSK (SEQ ID NO:43), QLATK2ohibuAAR (SEQ ID NO: 44), YQK2ohibuSTELLIR (SEQ ID NO: 45),EIAQDFK2ohibuTDLR (SEQ ID NO: 46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47),DNIQGITK2ohibuPAIR (SEQ ID NO: 48), K2ohibu TVTAMDVVYALK (SEQ ID NO:49), TVTAMDVVYALK2ohibuR (SEQ ID NO: 50), KpropK2ohibuAAKpropKpropPAGVR(SEQ ID NO: 51), KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51),KpropAAKpropK2ohibuPAGVR (SEQ ID NO: 52),K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 6. The method of claim 1, wherein the peptide consistsof an amino acid sequence selected from the group consisting ofDAVTYTEHAK2ohibuR (SEQ ID NO: 29), ASGPPVSELITK2ohibuAVAAS (SEQ ID NO:30), AVAASK2ohibuER (SEQ ID NO: 31), SGVSLAALK2ohibuK (SEQ ID NO: 32),K2ohibuALAAAGYDVEK (SEQ ID NO: 33), LGLK2ohibuSLVSK (SEQ ID NO: 34),propLGLK2ohibuSLVSKprop (SEQ ID NO: 34), SLVSK2ohibuGTLVQTK (SEQ ID NO:35), GTLVQTK2ohibuGTGASGSFK (SEQ ID NO: 36), NDEELNK2ohibuLLGK (SEQ IDNO: 37), PEPSK2ohibuSAPAPK (SEQ ID NO: 38), VLK2ohibuQVHPDTGISSK (SEQ IDNO: 39), LAHYNK2ohibuR (SEQ ID NO: 40), LLLPGELAK2ohibuHAVSEGTK (SEQ IDNO: 41), HAVSEGTK2ohibuAVTK (SEQ ID NO: 42), AVTK2ohibuYTSSK (SEQ ID NO:43), QLATK2ohibuAAR (SEQ ID NO: 44), YQK2ohibuSTELLIR (SEQ ID NO: 45),EIAQDFK2ohibuTDLR (SEQ ID NO: 46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47),DNIQGITK2ohibuPAIR (SEQ ID NO: 48), K2ohibu TVTAMDVVYALK (SEQ ID NO:49), TVTAMDVVYALK2ohibuR (SEQ ID NO: 50), KpropK2ohibuAAKpropKpropPAGVR(SEQ ID NO: 51), KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51),KpropAAKpropK2ohibuPAGVR (SEQ ID NO: 52),K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 7. A method for producing an isolated affinity reagentthat binds specifically to a protein or a fragment thereof comprising alysine when the lysine is 2-hydroxyisobutyrylated, wherein the isolatedaffinity reagent does not bind to the protein or fragment thereof whenthe lysine is not 2-hydroxyisobutyrylated, comprising screening aprotein library using a peptide comprising a 2-hydroxyisobutyrylatedlysine.
 8. The method of claim 7, wherein the binding of the isolatedaffinity reagent to the protein or a fragment thereof depends on the2-hydroxyisobutyrylated lysine and a surrounding peptide sequencethereof, and wherein the surrounding peptide sequence comprises at leasttwo residues on each of the N-terminal and C-terminal sides of the2-hydroxyisobutyrylated lysine.
 9. The method of claim 7, wherein thepeptide further comprises at least two amino acid residues on each ofthe N-terminal and C-terminal sides of the 2-hydroxyisobutyrylatedlysine.
 10. The method of claim 7, wherein the peptide comprises anamino acid sequence at least 50% identical to a sequence selected fromthe group consisting of DAVTYTEHAK2ohibuR (SEQ ID NO: 29),ASGPPVSELITK2ohibuAVAAS (SEQ ID NO: 30), AVAASK2ohibuER (SEQ ID NO: 31),SGVSLAALK2ohibuK (SEQ ID NO: 32), K2ohibuALAAAGYDVEK (SEQ ID NO: 33),LGLK2ohibuSLVSK (SEQ ID NO: 34), propLGLK2ohibuSLVSKprop (SEQ ID NO:34), SLVSK2ohibuGTLVQTK (SEQ ID NO: 35), GTLVQTK2ohibuGTGASGSFK (SEQ IDNO: 36), NDEELNK2ohibuLLGK (SEQ ID NO: 37), PEPSK2ohibuSAPAPK (SEQ IDNO: 38), VLK2ohibuQVHPDTGISSK (SEQ ID NO: 39), LAHYNK2ohibuR (SEQ ID NO:40), LLLPGELAK2ohibuHAVSEGTK (SEQ ID NO: 41), HAVSEGTK2ohibuAVTK (SEQ IDNO: 42), AVTK2ohibuYTSSK (SEQ ID NO: 43), QLATK2ohibuAAR (SEQ ID NO:44), YQK2ohibuSTELLIR (SEQ ID NO: 45), EIAQDFK2ohibuTDLR (SEQ ID NO:46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47), DNIQGITK2ohibuPAIR (SEQ ID NO:48), K2ohibu TVTAMDVVYALK (SEQ ID NO: 49), TVTAMDVVYALK2ohibuR (SEQ IDNO: 50), KpropK2ohibuAAKpropKpropPAGVR (SEQ ID NO: 51),KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51), KpropAAKpropK2ohibuPAGVR(SEQ ID NO: 52), K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 11. The method of claim 7, wherein the peptidecomprises an amino acid sequence selected from the group consisting ofDAVTYTEHAK2ohibuR (SEQ ID NO: 29), ASGPPVSELITK2ohibuAVAAS (SEQ ID NO:30), AVAASK2ohibuER (SEQ ID NO: 31), SGVSLAALK2ohibuK (SEQ ID NO: 32),K2ohibuALAAAGYDVEK (SEQ ID NO: 33), LGLK2ohibuSLVSK (SEQ ID NO: 34),propLGLK2ohibuSLVSKprop (SEQ ID NO: 34), SLVSK2ohibuGTLVQTK (SEQ ID NO:35), GTLVQTK2ohibuGTGASGSFK (SEQ ID NO: 36), NDEELNK2ohibuLLGK (SEQ IDNO: 37), PEPSK2ohibuSAPAPK (SEQ ID NO: 38), VLK2ohibuQVHPDTGISSK (SEQ IDNO: 39), LAHYNK2ohibuR (SEQ ID NO: 40), LLLPGELAK2ohibuHAVSEGTK (SEQ IDNO: 41), HAVSEGTK2ohibuAVTK (SEQ ID NO: 42), AVTK2ohibuYTSSK (SEQ ID NO:43), QLATK2ohibuAAR (SEQ ID NO: 44), YQK2ohibuSTELLIR (SEQ ID NO: 45),EIAQDFK2ohibuTDLR (SEQ ID NO: 46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47),DNIQGITK2ohibuPAIR (SEQ ID NO: 48), K2ohibu TVTAMDVVYALK (SEQ ID NO:49), TVTAMDVVYALK2ohibuR (SEQ ID NO: 50), KpropK2ohibuAAKpropKpropPAGVR(SEQ ID NO: 51), KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51),KpropAAKpropK2ohibuPAGVR (SEQ ID NO: 52),K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 12. The method of claim 7, wherein the peptideconsists of an amino acid sequence selected from the group consisting ofDAVTYTEHAK2ohibuR (SEQ ID NO: 29), ASGPPVSELITK2ohibuAVAAS (SEQ ID NO:30), AVAASK2ohibuER (SEQ ID NO: 31), SGVSLAALK2ohibuK (SEQ ID NO: 32),K2ohibuALAAAGYDVEK (SEQ ID NO: 33), LGLK2ohibuSLVSK (SEQ ID NO: 34),propLGLK2ohibuSLVSKprop (SEQ ID NO: 34), SLVSK2ohibuGTLVQTK (SEQ ID NO:35), GTLVQTK2ohibuGTGASGSFK (SEQ ID NO: 36), NDEELNK2ohibuLLGK (SEQ IDNO: 37), PEPSK2ohibuSAPAPK (SEQ ID NO: 38), VLK2ohibuQVHPDTGISSK (SEQ IDNO: 39), LAHYNK2ohibuR (SEQ ID NO: 40), LLLPGELAK2ohibuHAVSEGTK (SEQ IDNO: 41), HAVSEGTK2ohibuAVTK (SEQ ID NO: 42), AVTK2ohibuYTSSK (SEQ ID NO:43), QLATK2ohibuAAR (SEQ ID NO: 44), YQK2ohibuSTELLIR (SEQ ID NO: 45),EIAQDFK2ohibuTDLR (SEQ ID NO: 46), VTIMPK2ohibuDIQLAR (SEQ ID NO: 47),DNIQGITK2ohibuPAIR (SEQ ID NO: 48), K2ohibu TVTAMDVVYALK (SEQ ID NO:49), TVTAMDVVYALK2ohibuR (SEQ ID NO: 50), KpropK2ohibuAAKpropKpropPAGVR(SEQ ID NO: 51), KpropKpropAAK2ohibuKpropPAGVR (SEQ ID NO: 51),KpropAAKpropK2ohibuPAGVR (SEQ ID NO: 52),K2ohibuASGPPVSELITKpropAVAASK2ohibu (SEQ ID NO: 53),KpropASGPPVSELITKpropAVAASK2ohibuER (SEQ ID NO: 54),ALAAAGYDVEK2ohibuNNSR (SEQ ID NO: 55), IK2ohibuLGLK (SEQ ID NO: 56),propGILVQTK2ohibuGTGASGSFKprop (SEQ ID NO: 57),propK2ohibuAASGEAKpropPQAKprop (SEQ ID NO: 58),propAASGEAK2ohibuPQAKprop (SEQ ID NO: 59),propAASGEAKpropPQAK2ohibuKprop (SEQ ID NO: 60),propAK2ohibuKpropPAGAAKprop (SEQ ID NO: 61),propAKpropKpropPAGAAK2ohibuKpropPKprop (SEQ ID NO: 62),propKpropATGAATPK2ohibuKprop (SEQ ID NO: 63),propKpropAK2ohibuKpropPAAAAVTKprop (SEQ ID NO: 64),propKpropPAAAAVTK2ohibuKprop (SEQ ID NO: 65), KpropVAAKpropKpropK2ohibu(SEQ ID NO: 66), GK2ohibuQGGK2ohibuAR (SEQ ID NO: 67), K2ohibuGNYSER(SEQ ID NO: 68), DNK2ohibuK2ohibuTR (SEQ ID NO: 69), NDEELNK2ohibuLLGR(SEQ ID NO: 70), VTIAQGGVLPNIQAVLLPK2ohibuK (SEQ ID NO: 71),propPEPAK2ohibuSAPAPKprop (SEQ ID NO: 72), PEPAK2ohibuSAPAPK (SEQ ID NO:72), propPEPAKpropSAPAPKpropK2ohibuGSK (SEQ ID NO: 73),KpropAISK2ohibuAQKprop (SEQ ID NO: 74),AVTKpropAQK2ohibuKpropDGKpropKpropR (SEQ ID NO: 75),AVTKpropAQKpropK2ohibuDGKpropKpropR (SEQ ID NO: 75), K2ohibuESYSVYVYK(SEQ ID NO: 76), KESYSVYVYK2ohibuVLK (SEQ ID NO: 77),QVHPDTGISSK2ohibuAMGIMNSFVNDIFER (SEQ ID NO: 78), TK2ohibuQTAR (SEQ IDNO: 79), K2ohibuSTGGKacAPR (SEQ ID NO: 80), KpropSTGGK2ohibuAPR (SEQ IDNO: 80), K2ohibuQLATKacAAR (SEQ ID NO: 81), KQLATK2ohibuAAR (SEQ ID NO:81), KacQLATK2ohibuAAR (SEQ ID NO: 81), K2ohibuSAPATGGVKpropKpropPHR(SEQ ID NO: 82), KpropSAPATGGVK2ohibuKpropPHR (SEQ ID NO: 82), KohibuLPFQR (SEQ ID NO: 83), K2ohibuGGKacGLGKacGGAKacR (SEQ ID NO: 84),GKacGGK2ohibuGLGKacGGAKacR (SEQ ID NO: 85), GLGK2ohibuGGAKacR (SEQ IDNO: 86), GGKpropGLGKpropGGAK2ohibuR (SEQ ID NO: 87),GGK2ohibuGLGKacGGAKacR (SEQ ID NO: 87), GGKacGLGK2ohibuGGAKacR (SEQ IDNO: 87), RGGVK2ohibuR (SEQ ID NO: 88), GVLK2ohibuVFLENVIR (SEQ ID NO:89), KTVTAMDVVYALK2ohibuR (SEQ ID NO: 90), AGSAAK2ohibuASQSR (SEQ ID NO:91), K2ohibuETYSSYIYK (SEQ ID NO: 92), ETYSSYIYK2ohibuVLK (SEQ ID NO:93), IATEASK2ohibuLAAYNK (SEQ ID NO: 94), LILPGELAK2ohibuHAVSEGTR (SEQID NO: 95), FQK2ohibuSTELLIR (SEQ ID NO: 96), DSVTYTEHAK2ohibuR (SEQ IDNO: 97), K2ohibuTVTSLDVVYALK (SEQ ID NO: 98), TVTSLDVVYALK2ohibuR (SEQID NO: 99), GMGK2ohibuVGAK (SEQ ID NO: 100), K2ohibuSAPATGGIK (SEQ IDNO: 101), YQK2ohibuSTDLLIR (SEQ ID NO: 102), VTIMTK2ohibuDMQLAR (SEQ IDNO: 103), K2ohibuAPAAAAEK (SEQ ID NO: 104), KacAPAAAAEK2ohibuK (SEQ IDNO: 105), VLK2ohibuQVHPDVGISK (SEQ ID NO: 106), IALESSK2ohibuLVR (SEQ IDNO: 107), HAISEGTK2ohibuAVTK (SEQ ID NO: 108), AVTK2ohibuFSSSTN (SEQ IDNO: 109), TASSK2ohibuQVSR (SEQ ID NO: 110), AGGKacAGK2ohibuDSGK (SEQ IDNO: 111), AGGK2ohibuAGKacDSGK (SEQ ID NO: 111) and SAPAPK2ohibuKacGSK(SEQ ID NO: 112).
 13. An isolated affinity reagent produced according tothe method of claim
 1. 14. An isolated affinity reagent producedaccording to the method of claim
 2. 15. An isolated affinity reagentproduced according to the method of claim
 3. 16. An isolated affinityreagent produced according to the method of claim
 7. 17. An isolatedaffinity reagent produced according to the method of claim
 8. 18. Anisolated affinity reagent produced according to the method of claim 9.19. A method for detecting a 2-hydroxyisobutyrylated lysine in a proteinor a fragment thereof, comprising: (a) contacting the protein or afragment thereof with the isolated affinity reagent of claim 13, wherebythe isolated affinity reagent and the protein or a fragment thereofforms a binding complex, and (b) detecting the binding complex, whereinthe presence of the binding complex indicates the presence of a2-hydroxyisobutyrylated lysine in the protein or a fragment thereof. 20.A method for detecting a 2-hydroxyisobutyrylated lysine in a protein ora fragment thereof, comprising: (a) contacting the protein or a fragmentthereof with the isolated affinity reagent of claim 16, whereby theisolated affinity reagent and the protein or a fragment thereof forms abinding complex, and (b) detecting the binding complex, wherein thepresence of the binding complex indicates the presence of a2-hydroxyisobutyrylated lysine in the protein or a fragment thereof.